Multi-specific antibodies, capable of binding to different antigens (bispecific antibodies (bsAb), for example), are useful in clinical fields such as immunodiagnosis, immunotherapy, and diagnosis based on immunoassays. For example, multi-specific antibodies can be used for immobilizing enzymes used for enzyme immunoassays. In such cases, one arm of a multi-specific antibody is designed to bind to an epitope on an enzyme region that does not interfere with the enzyme reaction. The other arm is designed to bind to an immobilizing carrier, so that the enzyme is immobilized on the carrier via the antibody (Hammerling et al., J. Exp. Med. 128: 1461-1473 (1968)). In addition, it has been reported that multi-specific antibodies can be used for immunodiagnosis of a variety of diseases, including cancers (Songsivilai et al., Clin. Exp. Immunol. 79: 315-321 (1990)). For example, bispecific antibodies used for cancer diagnosis are designed such that one arm of the antibody recognizes a tumor-related antigen, and the other arm binds to a detectable marker (for example, Le Doussal et al., Int. J. Cancer Suppl. 7: 58-62 (1992); Le Doussal et al., J. Nucl. Med. 34: 1662-1671 (1993); Stickney et al., Cancer Res. 51: 6650-6655 (1991)).
Furthermore, in patients, multi-specific antibodies are known to be used for inducing cellular immune responses against pathogens or tumor cells (Segal and Snider, Chem. Immunol. 47: 179-213 (1989); Segal et al., Biologic Therapy of Cancer 2(4) De Vita et al. eds., J. B. Lippomcott, Philadelphia (1992) p. 1; Hsieh-Ma et al., Cancer Res. 52: 6832-6839 (1992); Weiner et al., Cancer Res. 53: 94-100 (1993)). Multi-specific antibodies can also be designed to induce T-cell-mediated cytotoxicity (Shalaby et al., J. Exp. Med. 175(1): 217-225 (1992); de Liji et al., “Bispecific Antibodies and Targeted Cellular Cytotoxicity”, Romet-Lemonne, Fanger and Segal eds., Lienhart (1991) p. 249; Clark et al., “Bispecific Antibodies and Targeted Cellular Cytotoxicity”, Romet-Lemonne, Fanger and Segal Eds. Lienhart (1991) p. 243; Kroesen et al., Cancer Immunol. Immunother. 37: 400-407 (1993); Kroesen et al., Br. J. Cancer 70: 652-661 (1994); Weiner et al., J. Immunol. 152: 2385-2392 (1994)). Moreover, multi-specific antibodies can be used as fibrinolytic agents or vaccination adjuvants, and also for treatment of infectious diseases (for example, targeting cells infected with HIV, influenza, trypanosomes, and such), delivering antitoxins to tumor cells, and bringing immune complexes to cell surface receptors (Fanger et al., as described above).
Conventionally, multi-specific antibodies were produced by methods such as (1) chemical coupling of different antibodies with distinct specificities using hetero-bifunctional linkers (Paulus, Behring Inst. Mitt., 78:118-132 (1985)); (2) fusion of hybridoma cells secreting different monoclonal antibodies (Milstein and Cuello, Nature 305: 537-539 (1983)); and (3) transfection of genes encoding light chains and heavy chains of different monoclonal antibodies (four genes) into mouse myeloma cells or other eukaryotic expression systems, followed by isolating monovalent portions with bispecificity (Zimmermann, Rev. Physiol. Biochem. Pharmacol. 105: 176-256 (1986); van Dijk et al., Int. J. Cancer 43: 944-949 (1989)).
Diabodies (Db) are bivalent antibody fragments constructed by gene fusion (Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO93/11161). Diabodies are dimers comprising two polypeptide chains, where each polypeptide chain comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) connected with a linker short enough to prevent interaction of these two domains, for example a linker of about five amino acids. The VL and VH domains encoded on the same polypeptide chain will form a dimer because the linker between the VL and VH is too short to form a single chain variable region fragment (scFv). Thus, the result is a diabody that comprises two antigen-binding sites. If the VL and VH domains directed against two different antigens (a and b) are expressed simultaneously as combinations of VLa-VHb and VLb-VHa connected with a linker of about five residues, they can be secreted as a bispecific diabody. Bispecific diabodies are a type of multi-specific antibody.